Doping Control Laboratory, AIT Seibersdorf Laboratories, Seibersdorf, Austria.
Drug Test Anal. 2009 Nov;1(11-12):494-504. doi: 10.1002/dta.97.
The detection of doping with MIRCERA (the brand name for Continuous Erythropoietin Receptor Activator, or CERA) is hampered by the limited excretion of the rather large molecule (approximately 60 kDa) in urine. Blood (serum, plasma) in combination with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) appears to be the ideal matrix for detecting all forms of doping with erythropoiesis-stimulating agents (ESAs) because the apparent molecular masses of ESAs are different from the mass of human serum erythropoietin (shEPO). While SDS-PAGE has proven the most sensitive method for the detection of doping with Dynepo, the sensitivity of SDS-PAGE for MIRCERA is drastically decreased. By exchanging the SDS for SARCOSYL (SAR) in the sample and running buffers the sensitivity problem was solved. SARCOSYL, a methyl glycine-based anionic surfactant, is only binding to the protein-part of MIRCERA but not to its polyethylene glycol (PEG)-chain, while SDS binds to both parts. In consequence, the monoclonal anti-EPO antibody (clone AE7A5) no longer interacts with the fully SDS-solubilized MIRCERA molecules. Only those molecules that contain SDS bound to the protein-chain are detected. Due to the inability of SARCOSYL to solubilize PEG-molecules, MIRCERA can be detected on SARCOSYL-PAGE with the same sensitivity as non-PEGylated epoetins. In a typical SAR-PAGE experiment, 200 microL of serum are used, which allows the direct detection of MIRCERA, recombinant epoetins (such as NeoRecormon, Dynepo, NESP), and shEPO in a single experiment and with high (i.e. femtogram) sensitivity.
米瑞替康（持续红细胞生成素受体激动剂或 CERA 的商品名）的检测受到相当大的分子（约 60 kDa）在尿液中排泄有限的阻碍。血液（血清、血浆）与十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）结合似乎是检测所有形式的促红细胞生成素刺激剂（ESA）兴奋剂的理想基质，因为 ESA 的表观分子量与人类血清促红细胞生成素（shEPO）的分子量不同。虽然 SDS-PAGE 已被证明是检测 Dynopo 兴奋剂最敏感的方法，但 MIRCERA 的 SDS-PAGE 灵敏度却大大降低。通过在样品和运行缓冲液中交换 SDS 为 SARCOSYL（SAR），解决了灵敏度问题。SARCOSYL 是一种基于甲基甘氨酸的阴离子表面活性剂，仅与 MIRCERA 的蛋白质部分结合，而不与聚乙二醇（PEG）链结合，而 SDS 则与两者都结合。因此，单克隆抗 EPO 抗体（克隆 AE7A5）不再与完全 SDS 溶解的 MIRCERA 分子相互作用。仅检测到含有 SDS 结合到蛋白质链的分子。由于 SARCOSYL 无法溶解 PEG 分子，因此可以在 SARCOSYL-PAGE 上以与非 PEG 化 epoetins 相同的灵敏度检测 MIRCERA。在典型的 SAR-PAGE 实验中，使用 200 μL 血清，可以在单次实验中以高（即飞克）灵敏度直接检测 MIRCERA、重组 epoetins（如 NeoRecormon、Dynepo、NESP）和 shEPO。
